Robert C. Burnett

Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes.

Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1–10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.

Histocompatibility testing of dog families with highly polymorphic microsatellite markers

The dog has served traditionally as a model for marrow and organ transplantation. A key component of any study of transplantation is histocompatibility typing of donors and recipients. Towards the development of a less expensive, more simplified typing system within canine families, a new highly polymorphic microsatellite marker for the canine Major Histocompatibility Complex class II region was isolated and characterized. In addition, we report on the application of class I and class II microsatellite-based markers for following the inheritance of the alleles within the canine analog of the human HLA loci, DLA, through multi-generation pedigree.

Distinct B-Cell and T-Cell Lymphoproliferative Disease Prevalence among Dog Breeds Indicates Heritable Risk

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor gamma chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using chi2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

Cutting Edge: The Acquisition of TLR Tolerance during Malaria Infection Impacts T Cell Activation

An effective immune response to infection requires control of pathogen growth while minimizing inflammation-associated pathology. During malaria infection, this balance is particularly important. Murine malaria is characterized by early production of proinflammatory cytokines, which declines in the face of continuing parasitemia. The mechanism by which this occurs remains poorly understood. In this study, we investigated the role of dendritic cells (DCs) in regulating pro- and anti-inflammatory cytokine responses. As malaria infection progresses, DCs become refractory to TLR-mediated IL-12 and TNF-α production, while increasing their ability to produce IL-10 and retaining the capacity for activation of naive T cells. IL-12-secreting DCs from early infection stimulate an IFN-γ-dominated T cell response, whereas IL-10-secreting DCs from later stages induce an IL-10-dominated T cell response. We suggest that phenotypic changes in DCs during Plasmodium yoelii infection represent a mechanism of controlling host inflammation while maintaining effective adaptive immunity.

USE OF (CA)N POLYMORPHISMS TO DETERMINE THE ORIGIN OF BLOOD CELLS AFTER ALLOGENEIC CANINE MARROW GRAFTING

We have used a polymerase chain reaction-based assay measuring polymorphic (CA)n repeats, a class of simple sequence repeats, to assess the success of allogeneic canine marrow transplants. Results were compared with those obtained with karyotype analysis of dividing cells in recipients that were sex mismatched with their marrow donors. Twenty recipients were conditioned for transplantation of genotypically DLA-identical littermate marrow by 450 cGy of total-body irradiation. In 2 recipients, results could not be compared, since either only cytogenetic or dinucleotide (CA)n marker data existed. Both dogs had autologous marrow recovery. In 15 of the remaining 18 recipients, complete agreement was found between the results obtained with dinucleotide (CA)n markers, cytogenetic studies, and granulocyte changes after transplantation. Seven of the 15 showed eventual autologous recovery, 6 displayed mixtures of host and donor cells, and 2 showed donor-type hematopoiesis. Two of the 18 dogs showed mixed chimerism with (CA)n markers and autologous recovery by cytogenetics, findings that may be related to differences in cells analyzed by the two techniques–i.e., all nucleated cells by (CA)n markers versus dividing cells by cytogenetics. In one additional recipient, results of marrow cytogenetics, granulocyte changes, and (CA)n markers were consistent with a successful allograft, while peripheral blood cytogenetics suggested autologous recovery, possibly the result of erroneous blood sampling. Polymerase chain reaction-based testing for dinucleotide repeat (CA)n polymorphisms, originally developed for genetic mapping in the dog, is useful and reliable when compared with cytogenetic studies, in assessing the success of allogeneic marrow transplants in dogs.

Multicenter Prospective Trial of Hypofractionated Radiation Treatment, Toceranib, and Prednisone for Measurable Canine Mast Cell Tumors

BACKGROUND Mast cell tumors (MCT) are common cutaneous tumors in dogs and when not amenable to surgical excision can present a therapeutic challenge. New treatment protocols for unresectable MCT are needed. HYPOTHESIS The combination of toceranib, prednisone, and hypofractionated radiation treatment (RT) will be well tolerated and efficacious. ANIMALS Seventeen client-owned dogs with measurable MCT amenable to RT. METHODS Prospective clinical trial. All dogs received prednisone, omeprazole, diphenhydramine, and toceranib. Toceranib was administered for 1 week before initiating RT, consisting of 24 Gy delivered in 3 or 4 fractions. RESULTS On an intent-to-treat basis, the overall response rate was 76.4%, with 58.8% of dogs achieving a complete response and 17.6% a partial response. The median time to best response was 32 days, and the median progression-free interval was 316 days. The overall median survival time was not reached with a median follow-up of 374 days. The most common toxicoses were gastrointestinal and hepatic. CONCLUSIONS AND CLINICAL IMPORTANCE The combination of hypofractionated RT, toceranib, and prednisone was tolerated and efficacious in the majority of dogs. Response rates and durations were higher than those reported for toceranib as a single-agent treatment for MCT. This combination is a viable treatment option for unresectable MCT.

A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma

BACKGROUND Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor-initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B-cell lymphoma have not been conclusively identified. HYPOTHESIS/OBJECTIVES Tumor cells with a progenitor phenotype exist in B-cell lymphoma, reflecting a hierarchical organization. ANIMALS Twenty-eight client-owned dogs with previously untreated B-cell lymphoma and 6 healthy dogs. METHODS This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B-cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL-2Rγ(-/-) mice was adapted to expand and serially transplant primary canine B-cell lymphoma. RESULTS LPCs were expanded in lymph nodes from 28 dogs with B-cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B-cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. CONCLUSIONS AND CLINICAL IMPORTANCE These results suggest the presence of a hierarchy of tumor cells in B-cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.

CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

Abstract Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies.

Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor.

BackgroundMast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population.ResultsThe canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOC-resistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp.ConclusionsThis study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance.

Cloning and tissue expression of the equine transferrin receptor.

BACKGROUND Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses. OBJECTIVE This study was conducted to clone and sequence the eTfR gene and measure expression levels using quantitative real-time PCR and immunohistochemical (IHC) staining. METHODS Total RNA from equine bone marrow was used to produce cDNA. The eTfR gene was amplified using pooled gene-specific primers, and PCR products were sequenced. Rapid amplification of cDNA ends was used to obtain the first 22 nucleotides of the coding sequence. Quantitative PCR was performed using eTfR gene-specific primers, and IHC staining was used to localize TfR protein expression. RESULTS The deduced amino acid (aa) sequence (767 aa) of the eTfR was 75-83% identical with sequences of the receptor in several other mammals. As in people and rats, eTfR mRNA expression was highest in the bone marrow, and distribution in other tissues was also similar. CONCLUSION The eTfR gene is similar to that of other mammals in structure and expression levels. We hypothesize that it is also similar in function and that, following development of an immunoassay, determining sTfR concentrations will be useful for identifying the regenerative response in anemic horses.